Neuropathology Evaluation in Juvenile Toxicity Studies in Rodents: Comparison of Developmental Neurotoxicity Studies for Chemicals With Juvenile Animal Studies for Pediatric Pharmaceuticals

The developmental neuropathology examination in juvenile toxicity studies depends on the nature of the product candidate, its intended use, and the exposure scenario (eg, dose, duration, and route). Expectations for sampling, processing, and evaluating neural tissues differ for developmental neurotoxicity studies (DNTS) for chemicals and juvenile animal studies (JAS) for pediatric pharmaceuticals. Juvenile toxicity studies typically include macroscopic observations, brain weights, and light microscopic evaluation of routine hematoxylin and eosin (H&E)-stained sections from major neural tissues (brain, spinal cord, and sciatic nerve) as neuropathology endpoints. The DNTS is a focused evaluation of the nervous system, so the study design incorporates perfusion fixation, plastic embedding of at least one nerve, quantitative analysis of selected brain regions, and sometimes special neurohistological stains. In contrast, the JAS examines multiple systems, so neural tissues undergo conventional tissue processing (eg, immersion fixation, paraffin embedding, H&E staining only). An “expanded neurohistopathology” (or “expanded neuropathology”) approach may be performed for JAS if warranted, typically by light microscopic evaluation of more neural tissues (usually additional sections of brain, ganglia, and/or more nerves) or/and special neurohistological stains, to investigate specific questions (eg, a more detailed exploration of a potential neuroactive effect) or to fulfill regulatory requests.

Regulatory Perspectives on Juvenile Animal Toxicologic Pathology

The Society of Toxicologic Pathology’s Annual Virtual Symposium (2021) included a session on "Regulatory Perspectives on Juvenile Animal Toxicologic Pathology." The following narrative summarizes the key concepts from the four talks included in this symposium session chaired by Drs Deepa Rao and Alan Hoberman. These encompass an overview of various global regulations impacting the conduct of juvenile animal studies in pharmaceutical drug development and chemical toxicity assessments in a talk by Dr Alan Hoberman. Given the numerous regulatory guidances and legal statutes that have covered the conduct of juvenile animal studies and the recent harmonization of these guidances for pharmaceuticals, Dr Paul Brown provided an update on the harmonization of these guidances for pharmaceuticals, in the recently finalized version of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S11 guidance document, “Nonclinical Safety Testing in Support of Development of Pediatric Medicines.” The first two talks on regulations were followed by two talks focused on an evaluation of the postnatal development of two major organ systems relevant in juvenile animals. Dr Aurore Varela covered study design and endpoints impacting the skeletal system (bone), while Dr Brad Bolon presented a talk on the study design and conduct of neuropathology evaluations for the developing nervous system.

Analysis of seroprevalence in target wildlife during the oral rabies vaccination programme in Lithuania

Background Rabies vaccination of wildlife carnivores is a powerful tool to prevent, control and eliminate rabies. The presence of neutralizing rabies antibodies in blood is considered a reliable indicator of adequate vaccination. The main purpose of the present study was to analyze the seroprevalence of specific antibodies in target populations of Lithuanian red fox (RF) and raccoon dog (RD) during the oral rabies vaccination (ORV) campaigns during the 2010–2019 period. Results Over the ten-year period, 7,261 RF and 2,146 RD sera samples were collected post-mortem in field conditions and tested using a commercial standardized enzyme-linked immunosorbent assay (ELISA) kit in Lithuania. In the ORV spring and autumn vaccination periods, 31.8% (20.3–43.4 95% CI – 95% confidence interval) and 31.7% (21.2–42.1 95% CI) of RF, and 34.1% (22.5–45.7 95% CI) and 34.7% (22.7–46.7 95% CI) of RD sera samples, respectively, were identified as ELISA-positive (seroconversion ≥ 0.5 EU/mL—Equivalent Units per Millilitre). The seroprevalence analysis in adult/ juvenile animal subpopulations indicated that 34.9% (27.2–42.5 95% CI) and 29.2% (20.3–37.9 95% CI) of RF, and 35.6% (25.2–46.0 95% CI) and 30.6% (20.2–40.9 95% CI) of RD sera samples, respectively, were identified as ELISA-positive (seroconversion ≥ 0.5 EU/mL). Statistically strong determinate correlations (r) between the serological results (pos.%) in RF adult/juvenile animal subpopulations (r = 0.937) and between RF and RD positive seroconvert (pos.%) sera samples during the spring vaccinations (r = 0.864) were demonstrated. In different ORV periods, 14–29% of RF and 7–25% of RD sera samples were identified as ELISA-negative (seroconversion < 0.5 EU/mL), but with low (0.125 < 0.49 EU/mL) antibody (Abs) titres. Conclusions The 2010–2019 ORV programme has been an effective tool in both RF and RD populations in Lithuania. The rabies-free status of Lithuania was self-declared in 2015 with only three rabies cases identified in buffer zones since then. The percentage of ELISA-positive serum samples (seroconversion ≥ 0.5 EU/mL) during the different periods of vaccination was similar in RF and RD populations—32% and 34% respectively. The identified seroconversion average of 21.5% in RF and 16% in RD sera samples were officially identified as ELISA-negative (seronversion < 0.5 EU/mL), but with low 0.125 < 0.49 EU/mL Abs titres. That low, but positive seroconversion participated in the formation of populations overall immune status and can influence the interpretation of oral vaccination efficacy.

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country

Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

Evaluation of Juvenile Animal Studies for Pediatric CNS-Targeted Compounds: A Regulatory Perspective

Central nervous system (CNS)-targeted products are an important category of pediatric pharmaceuticals. In view of the significant postnatal maturation of the CNS, juvenile animal studies (JAS) are performed to support pediatric development of these new medicines. In this project, the design and results of juvenile toxicity studies from 15 drug compounds for the treatment of neurologic or psychiatric conditions were analyzed. Studies were conducted mostly in rats; sometimes in addition in dogs and monkeys. The study design of the pivotal JAS was variable, even for compounds with a similar therapeutic indication. Age of the juvenile animals was not consistently related to the starting age of the intended patient population. Of 15 compounds analyzed, 6 JAS detected more severe toxicities and 6 JAS evidenced novel CNS effects compared to their adult counterparts. The effects of CNS on acoustic startle and learning and memory were observed at high dosages. Reversibility was tested in most cases and revealed some small effects that were retained or only uncovered after termination of treatment. The interpretation of the relevance of these findings was often hampered by the lack of matching end points in the adult studies or inappropriate study designs. Detailed clinical observation and motor activity measures were the most powerful end points to detect juvenile CNS effects. The need for more detailed behavioral examinations in JAS, for example, on learning and memory, should, therefore, be decided upon on a case-by-case basis, based on specific concerns in order to avoid overloading the studies.

CDER Experience With Juvenile Animal Studies for CNS Drugs

A survey was undertaken to evaluate juvenile animal studies conducted for drug applications reviewed by the Center for Drug Evaluation and Research between 2009 and 2014. Some conclusions about the nonclinical pediatric safety assessment based on studies performed in support of central nervous system–active compounds are presented here. A total of 44 completed studies from 32 New Drug Applications submitted to the Divisions of Psychiatry and Neurology Products were evaluated. Data on animal species and age range used, endpoints evaluated, and outcomes included in labeling were analyzed. Of the drugs evaluated, all but one had studies conducted in rats. In some cases, a second study in a nonrodent species (dog) was also conducted. Indices of growth and development and standard general toxicity parameters were included in all of the studies. Expanded neurohistopathology evaluations, bone mineral density measurements, and reproductive and neurobehavioral functional assessments were also generally carried out. A variety of neurological and neurobehavioral tests were employed. In the majority of rat studies, the potential for long-term cognitive impairment was evaluated using a complex water maze. Juvenile animal studies provided safety information considered relevant to drug use in children and that was included in labeling for 78% of the applications surveyed. The most commonly reported findings in labeling were for neurobehavioral effects, including changes in locomotor activity, auditory startle habituation, and learning and memory. Of the studies described in labeling with neurobehavioral effects, 54% found these effects to be persistent and to provide evidence of developmental neurotoxicity.